It was 50 years ago that Avery, MacLeod, and McCarty published their classic paper reporting that DNA was the material inducing the transformation of capsule types in the gram positive bacterium Streptococcus pneumoniae. Although the role of capsules in virulence has been well documented, the biosynthesis of capsular polysaccharides by this organism has received little attention from the research community over the last several decades. Almost nothing is known about the pathways, enzymes and mechanisms involved in the synthesis of these polysaccharides. Therefore, the long term objective of this research program is to develop a thorough understanding of the multi-enzyme systems and the molecular mechanisms that are involved in the biosynthesis of the capsular polysaccharides of this organism. Since more than 80 different serotypes of pneumococcal polysaccharides have been identified, the proposed studies will be limited to several specific capsule types for which the sugar compositions and structures, as well as the genetics, have been well established. Specific aims of the proposal are to: a) establish the overall pathways involved in the synthesis of specific pneumococcal polysaccharides; b) purify and characterize glycosyltransferases and polymerases involved in their biosynthesis; c) clone genes encoding the glycosyltransferases/polymerases, and prepare high expression systems as a source of enzymes for purification and characterization, d) investigate the effects of the phospholipid environment on enzyme activities; e) investigate factors that might serve to control or regulate enzyme activities in a phospholipid environment and; f) identify the catalytic mechanisms of the glycosyltransferases. Results obtained from the proposed research will provide significant new insight about the biosynthesis of the capsular polysaccharides of gram positive bacteria. The proposed research will take advantage of molecular cloning techniques, as well as recently developed methodologies for the purification and reconstitution of membrane glycosyltransferases, to provide new insight into the biosynthesis of bacterial capsules. The information obtained from these studies may potentially be useful in the long term for developing strategies for preventing or attenuating pneumococcal infections.